猪源CCL25重组蛋白表达及其生物学活性分析

doi:10.11843/j.issn.0366-6964.2025.11.042

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5864-5874.doi: 10.11843/j.issn.0366-6964.2025.11.042

猪源CCL25重组蛋白表达及其生物学活性分析

曹秋霞¹^,²(), 颜可欣², 程振孔², 卞贤宇², 王传红², 李素芬², 张雪寒², 范宝超², 郭容利², 杨珊珊²^,*(), 王晓杜¹^,*(), 李彬¹^,²^,*()

1. 浙江农林大学动物医学院, 杭州 311300
2. 江苏省农业科学院兽医研究所, 南京 210014

收稿日期:2025-01-21 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 杨珊珊,王晓杜,李彬 E-mail:1161824097@qq.com;yangshanshan@caas.cn;xdwang@zafu.edu.cn;libinana@126.com
作者简介:曹秋霞(1999-)，女，浙江天台人，硕士生，主要从事动物腹泻病感染机制研究，E-mail：1161824097@qq.com
基金资助:
国家重点研发计划项目(2023YFD1800302)；江苏省农业科技自主创新资金项目(CX(24)3071)；江苏省基础研究专项资金(BK20241180)；河北省省级科技计划资助(21322401D)；国家自然科学基金项目(32402904)

Expression and Biological Activity Analysis of Porcine CCL25 Recombinant Protein

CAO Qiuxia¹^,²(), YAN Kexin², CHENG Zhenkong², BIAN Xianyu², WANG Chuanhong², LI Sufen², ZHANG Xuehan², FAN Baochao², GUO Rongli², YANG Shanshan²^,*(), WANG Xiaodu¹^,*(), LI Bin¹^,²^,*()

1. College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Received:2025-01-21 Online:2025-11-23 Published:2025-11-27
Contact: YANG Shanshan, WANG Xiaodu, LI Bin E-mail:1161824097@qq.com;yangshanshan@caas.cn;xdwang@zafu.edu.cn;libinana@126.com

摘要/Abstract

摘要：

本研究旨在表达鉴定重组猪源细胞因子CCL25蛋白功能及制备针对CCL25的兔源多克隆抗体。获取去除跨膜区的CCL25氨基酸序列，构建重组载体并在大肠杆菌表达系统中表达，镍柱纯化后利用SDS-PAGE、Western blot及ELISA鉴定蛋白表达；利用细胞增殖、划痕试验、Transwell培养等试验，通过流式细胞术、实时荧光定量等技术验证蛋白生物活性。蛋白采用皮下多点注射方法免疫新西兰大白兔，制备兔多克隆抗体。通过间接ELISA、共聚焦及Western blot验证多克隆抗体的功能与效价。结果显示：SDS-PAGE、Western blot证实蛋白可溶性表达，细胞增殖、划痕试验、Transwell培养等试验结果显示，CCL25促进IPEC-J2细胞生长，使其向空白区域迁移，上调细胞AKT和p-AKT₄₇₃表达量，并明显促进猪外周血淋巴细胞的细胞趋化。同时，CCL25抑制LPS诱导的IL-8、caspase-3表达以及7-AAD⁺死亡细胞比例。除此之外，本研究成功制备出效价高、特异性良好的多克隆抗体。原核表达纯化获得CCL25蛋白并初步鉴定CCL25蛋白具有促进细胞增殖、迁移、趋化以及抗炎抗凋亡等生物学功能，为CCL25的后续功能研究提供了有效的实验工具和基础数据。

关键词: CCL25, 原核表达, 蛋白纯化, 功能鉴定, 多克隆抗体

Abstract:

The objective of this research is to investigate the function of recombinant porcine chemokine CCL25 protein and develop polyclonal antibodies targeting CCL25 from rabbits. The amino acid sequence of truncated CCL25 without the transmembrane region was obtained, a recombinant vector was constructed, and the protein was expressed in Escherichia coli expression system. Protein expression was confirmed by SDS-PAGE, western blot, and ELISA after nickel column purification. The biological activity of the protein was validated through cell proliferation assays, scratch tests and Transwell cultures using flow cytometry and real-time fluorescence quantification. New Zealand white rabbits were immunized subcutaneously with the protein to produce polyclonal antibodies. The functionality and titer of the polyclonal antibodies were confirmed using indirect ELISA, confocal microscopy, and western blot. The soluble expression of the protein was verified by SDS-PAGE and western blot, and the target protein was obtained after purification. Experimental results demonstrated that CCL25 promoted the growth and migration of IPEC-J2 cells, upregulated cell AKT and p-AKT₄₇₃ expression, and significantly enhanced chemotaxis of porcine peripheral blood lymphocytes. Additionally, CCL25 inhibited LPS-induced IL-8 and caspase-3 expression, as well as 7-AAD⁺ cell death ratio. In addition, this study successfully prepared polyclonal antibodies with high titer and good specificity. The CCL25 protein was obtained by prokaryotic expression and purification. It was preliminarily identified that CCL25 protein had biological functions such as promoting cell proliferation, migration, chemotaxis, anti-inflammation and anti-apoptosis, which provided an effective experimental tool and basic data for the subsequent functional study of CCL25.

Key words: CCL25, prokaryotic expression, protein purification, functional identification, polyclonal antibody

中图分类号:

S852.4

曹秋霞, 颜可欣, 程振孔, 卞贤宇, 王传红, 李素芬, 张雪寒, 范宝超, 郭容利, 杨珊珊, 王晓杜, 李彬. 猪源CCL25重组蛋白表达及其生物学活性分析[J]. 畜牧兽医学报, 2025, 56(11): 5864-5874.

CAO Qiuxia, YAN Kexin, CHENG Zhenkong, BIAN Xianyu, WANG Chuanhong, LI Sufen, ZHANG Xuehan, FAN Baochao, GUO Rongli, YANG Shanshan, WANG Xiaodu, LI Bin. Expression and Biological Activity Analysis of Porcine CCL25 Recombinant Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5864-5874.

图/表 8

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基因 Gene	序列(5′→3′) Sequence
caspase-3	F: TGGGATTGAGACGGACAGTG；R: CGCTGCACAAAGTGACTGGA
IL-8	F: TGCACTTACTCTTGCCAGAACTG；R: CAAACTGGCTGTTGCCTTCTT
ACTIN	F: GGACTTCGAGCAGGAGATGG；R: AGGAAGGAGGGCTGGAAGAG

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猪源CCL25重组蛋白表达及其生物学活性分析

Expression and Biological Activity Analysis of Porcine CCL25 Recombinant Protein

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